Ezrin is a multifunctional protein that connects the actin cytoskeleton to extracellular matrix through transmembrane proteins. High ezrin expression is associated with lung metastasis and poor survival rates in cancer.
Osteosarcoma (OS) is the most common type of primary bone cancer in children and adolescents. The pathogenesis underlying the disease has been difficult to establish due to its heterogenous histology and complex etiology. Treatment of the localized disease has improved with introduction of neoadjuvant chemotherapy, increasing the 5-year survival to 60-70%. However, 5-year survival of patients with metastasis at diagnosis decreases to 30% (Zhang, P., et al., Clin. Cancer Res., 14, 2962-2969 (2008); Rosen G., et al., Cancer, 49, 1221-1230 (1982); Ferrari, S. & Palmerini, E., Curr. Opin. Oncol., 19, 341-346 (2007); Bacci, G., et al., Cancer, 106, 1154-1161 (2006)). In OS, the predominatnt site of recurrence and the main cause of death are pulmonary metastases (Dunn, D. & Dehner, L. P., Cancer, 40, 3054-3064 (1977)). Targeting the underlying molecular events that lead to metastasis could provide dramatic benefits for the treatment of patients with poor prognosis.
Ezrin is a member of the ERM (Ezrin/Radixin/Moesin) family of proteins and is conserved through evolution both structurally and functionally (Fievet, B., et al., Biochim. Biophys. Acta, 1773, 653-660 (2007)). By regulating membrane-cytoskeleton complexes, it plays key roles in normal cellular processes like maintenance of membrane dynamics, survival, adhesion, motility, cytokinesis, phagocytosis and integration of membrane transport with signaling pathways (Bretscher, A., et al., Nat. Rev. Mol. Cell. Biol., 3, 586-599 (2002)). Both in vivo and in vitro studies show ezrin function is actively regulated by its conformational changes (Fievet, B., et al., Biochim. Biophys. Acta, 1773, 653-660 (2007)). Ezrin exists in an inactive conformation, in which the membrane and actin binding sites are masked by intramolecular interaction of the N-terminal and the last 100 amino acids of the long Carboxy terminal domains (Gary, R. & Bretscher, Mol. Biol. Cell, 6, 1061-1075 (1995)). In its active-open confirmation, it functions as a crosslinker between the plasma membrane and the cortical cytoskeleton. Two factors are reported to be involved in this conformational transition, binding of N-terminal domain to the phosphotidylinositol 4,5 biphosphates (PIP2) and phosphorylation of a conserved threonine at residue 567 (T567) in the F-actin binding site (Fievet, B., et al., Biochim. Biophys. Acta, 1773, 653-660 (2007)). Several serine/threonine kinases, Rho kinase (ROCK), protein kinase C-alpha (PKCα) and MST4, are reported to be important for T567 phosphorylation (Matsui, T., et al. J. Cell Biol., 140, 647-657 (1998); Ren, L., et al. Oncogene, 28, 792-802 (2009); Ten Klooster, J. P., et al. Dev. Cell, 16, 551-562 (2009)). In its active form, ezin can interact with membrane proteins either directly or through adaptor proteins. It binds to adhesion related proteins with single transmembrane domains such as CD43, CD44, CD95, ICAM-1, -2, -3 and PA.2.26 antigen directly through their cytoplasmic tails (Louvet-Vallee, S., Biol. Cell, 92, 305-316 (2000)), which modulates cell motility and cellular morphology (Legg, J. W. & Isache, Curr. Biol., 8, 705-708 (1998)). Ezrin binding to adaptor proteins such as EBP50/NHE-RF and E3KARP regulates the activity of ion transporters, endocytosis of plasma membrane proteins and interaction of F-actin to specific plasma membrane domains (Bretscher, A., et al., Nat. Rev. Mol. Cell. Biol., 3, 586-599 (2002)). In addition to plasma membrane proteins, ezrin associates with cytoplasmic signaling proteins and is involved in several signaling pathways, such as but not limited to Rho and PI3K/Akt pathways (Gautreau, A., et al., Proc. Natl. Acad. Sci. USA, 96, 7300-7305 (1999); and Hirao, M., et al. J. Cell Biol., 135, 37-51 (1996)). Ezrin can modulate these pathways at both upstream and the downstream levels.
Accumulating evidence from experimental mouse models, as well as canine and human patients validate that ezrin is a key factor in metastases. In this study, we identified that the compounds described herein directly interact with ezrin and inhibit its biological function in multiple assays.